1. Field of the Invention
This application is a continuation-in-part patent application of pending non-provisional patent application U.S. Ser. No. 12/462,959, filed on Aug. 12, 2009. This continuation-in-part patent application claims the benefit of prior non-provisional patent application U.S. Ser. No. 12/462,959, filed on Aug. 12, 2009, which patent application is hereby incorporated into this continuation-in-part patent application in its entirety by reference herein, including all drawings, all seed and/or other deposits made with the American Type Culture Collection (ATCC) and all sequence listings.
The present invention is directed to unique methods for producing plants, plant materials and seeds that very advantageously have enhanced production and consumer traits, such as an improved vigor and hardiness of the seeds, an enhanced seedling emergence and plant growth in both warm and cold soils (i.e., in different types of climates and at different temperatures), combined with a superior taste quality and kernel pericarp tenderness, and to improved plants, plant materials and seeds that are produced in accordance with these methods, or that have such traits. The invention provides a novel approach for combining a unique assembly of genetic traits in plants, plant materials and seeds, using double, triple and other allelic combinations, resulting in enhanced growing potential along with desirable consumer qualities.
2. Background
Maize
Corn (maize) is one of the most diverse grain crops that is present in nature, and there are a number of different types of corn, which are generally classified by characteristics of their kernel endosperm. The most common types of corn include flint, flour, dent, pop, sweet, waxy and pod. The physical appearance of each kernel type is determined by its endosperm pattern, quality and quantity.
Sweet corn is a kind of corn plant that is classified as Zea mays, var. rugosa, and that has white, yellow or bi-colored kernels that are sweet when they are in the immature milky stage as a result of having a high sugar content. Higher levels of sugar in the sweet corn kernels result in a lower osmotic potential, causing greater water uptake into the kernels. Sweet corn is typically eaten by human beings as a vegetable, either directly from the maize cob, or by having the sweet kernels removed from the cob, and is a major vegetable crop that is grown all over the world primarily for fresh consumption, rather than as animal feed or for flour production.
Sweet corn occurs as a spontaneous mutation in field corn, and can be the result of naturally-occurring mutations in the genes that control conversion of sugar to starch inside the endosperm of the corn kernel. Unlike field corn varieties, which are intended for livestock, and are typically harvested when their kernels are dry and fully mature (at the dent stage), sweet corn is typically picked when it is immature (at the milk stage), and eaten as a vegetable, rather than as a grain. Because the process of maturation involves converting sugar into starch, sweet corn typically stores poorly, and must be eaten in a fresh, canned or frozen manner before the kernels become tough and/or starchy. Following harvest, or if left on the stalk too long, sucrose in standard sweet corn becomes rapidly converted to starch. Kernels can lose as much as 50% of their sucrose at room temperature at around 24 hours after harvest.
Open pollinated (non-hybrid) varieties of white sweet corn started to become widely available in the United States in the 19th century. Two of the most enduring varieties, which are still available today, are Country Gentleman (a Shoepeg corn with small, white kernels in irregular rows) and Stowell's Evergreen. Sweet corn production in the 20th century was influenced by the following key developments:                (i) hybridization, which allowed for more uniform maturity, improved quality and disease resistance; and        (ii) identification of the separate gene mutations that are responsible for sweetness in corn, and the ability to breed varieties based on these characteristics, for example:                    su (normal sugary);            se (sugary enhanced); and            sh2 (shrunken-2).There are currently hundreds of varieties of sweet corn, with more varieties continuously being developed.                        
The fruit of the sweet corn plant is the corn kernel, and the ear consists of a collection of kernels on the cob. Because corn is a monocot, there is always an even number of rows of kernels. The ear is covered by tightly wrapped leaves (the husk).
Sweet corn has significant antioxidant activity, which can reduce the possibilities of developing heart disease and/or cancer. It also releases increased levels of ferulic acid, which provides additional health benefits.
There are several known genetic mutations that are responsible for the various types of sweet corn. Early varieties were the result of the mutant su1 (sugary-1) allele. Conventional su1 varieties contain about 5-10% sugar by weight.
Varieties of sweet corn that contain the shrunken-2 (sh2) gene typically produce higher than normal levels of sugar, and have a longer shelf life, in comparison with conventional sweet corn, and are frequently referred to as supersweet varieties. One specific gene in sweet corn, the shrunken-2 (sh2) gene, causes the mature corn kernel to dry and shrivel as it matures past the milky stage, which is an undesirable trait for seedling germination, early emergence and plant growth. The endosperm of conventional sh2 sweet corn kernels store less amounts of starch, and from about 4 to about 10 times more sugar, than conventional su1 sweet corn. This has permitted the long-distance shipping of sweet corn, and has enabled manufacturers to can sweet corn without adding extra sugar or salt to it.
The third gene mutation is the se1 (sugary enhanced-1) allele, which is incorporated in the genome of Everlasting Heritage varieties. Conventional sweet corn varieties with the se1 alleles typically have a longer storage life, and contain from about 12% to about 20% sugar (i.e., a much higher sugar level in comparison with the conventional su1 varieties).
All of the alleles that are responsible for sweet corn are recessive, so sweet corn must generally be isolated from any field corn varieties that release pollen at the same time. The endosperm develops from genes from both parents (male and female), and kernels will generally be tough and starchy.
Maize was first classified according to the carbohydrate that is stored in its endosperm. The most distinguishable sugar component that is present in sweet corn is sucrose, which accounts for the vast majority of its sweetness differentiation. (Abbott and Cobb, Inc., Plant Protection No. 9600094 (1998).) The reducing free sugars, glucose and fructose, are present in sweet corn in significantly lower levels. These reducing sugars primarily result from the natural hydrolysis of sucrose.
Most commercially-available sweet corns are based upon single recessive alleles that alter carbohydrate composition of the endosperm. Until about thirty years ago, sweet corn was typically defined by the su1 mutant allele located on chromosome four. Although the primary difference between sweet corn and field corn is historically due to a single gene, there are many other genes, both exhibiting major and minor effects that differentiate the two classifications of corn. These genes affect the overall eating quality of the corn (flavor, texture, tenderness and the like) along with the appearance of the kernel, ear and plant, as well as the viability of the seed. Sweet corn is differentiated mainly from other types of corn, i.e., flint, flour, dent, waxy, pop, and the like, by the presence of genes that typically alter endosperm starch synthesis and its use as a vegetable for human consumption.
Many of the endosperm mutant genes in maize (and in other crop plants) that are presently being used commercially in different sweet corn varieties, and affect endosperm carbohydrate synthesis, are listed in Table 1 below. These endosperm mutations all are believed to affect starch synthesis during kernel development, and have been characterized and mapped in maize.
TABLE 1Sweet Corn Endosperm Mutant Genes Used CommerciallyGeneGene SymbolChromosomeAmylose-extenderae5Brittlebt5Brittle-2bt24Dulldu10Shrunken-2sh23Sugary-1su14Sugary enhancer-1se12Waxywx9Of the endosperm mutants that are listed in Table 1, the mutants that are most widely used commercially are sugary-1 (su 1), sugary enhancer-1 (se1) and shrunken-2 (sh2).
Endosperm Mutants
Sweet corn eating quality is typically largely determined by its sweetness which, in turn, is affected by the amount of sugar and starch that are present in the endosperm of its kernels. The major and, therefore, most distinguishable sugar component in sweet corn is sucrose, which accounts for most of its sweetness differentiation. The reducing sugars, glucose and fructose, are typically present in sweet corn in significantly lower levels in comparison with sucrose. These reducing sugars typically result from the natural hydrolysis of sucrose. Sugary-1 (su1), sugary enhancer-1 (se1), and shrunken-2 (sh2) mutant alleles have been determined to markedly increase sugar content, and decrease starch levels, in sweet corn in comparison with corn that does not include one or more of these mutant alleles in its genome.
Another determining characteristic of sweet corn is that its kernels (seeds) tend to be markedly more tender in comparison with other types of corn. Significant degrees of tenderness levels may occur within sweet corn varieties expressing one or more of the above three (or other) endosperm mutant alleles. Elevated sugar levels and increased kernel pericarp tenderness that typically occur in these varieties generally allows for extended holding abilities or shelf lives. An extended holding ability or shelf life, both on and off of a corn plant (or other production or distribution facility), is generally extremely important when considering shipping sweet corn varieties over potentially long geographical distances to market, for example, across the United States, or from one country to another country.
Currently, for reasons related to elevated sugar and pericarp tenderness levels, the shrunken-2 (sh2) sweet corn class is the most popular type of sweet corn commercially available. However, as is described hereinbelow, such class has some very significant disadvantages, which are overcome by the processes and products of the present invention.
Su1 (Sugary-1) Mutant Gene
Sweet corn in its original form differs from field corn primarily by the presence of a single recessive mutant gene, sugary-1 (su1). The recessive sugary-1 (su1) genotype that is present in sweet corn has an effect of retarding (significantly slowing) a normal conversion of sugar into starch during endosperm development, which very desirably results in a sweet taste, rather than in a starchy taste. This gene has been cloned and mapped to the short arm of chromosome 4 in sweet corn, and its genomic sequence and amino acid sequence translation are set forth in U.S. Pat. No. 5,912,413 entitled “Isolation of the SU1 Starch Debranching Enzyme, the Product of the Maize Gene Sugary 1,” and herein.
The sugary (su1) gene encodes a Class II starch debranching enzyme that is active in cellular plastids. It is an isoamylase that hydrolyzes the α-(1,6) branch linkages in starch during starch synthesis. (J A Shultz et al., “Current Models for Starch Synthesis and the Sugary Enhancer 1 (se1) Mutation in Zea mays,” Plant Physiology and Biochemistry 42(6), 457-464 (2004).)
The sugary-1 (su1) gene confers a moderate increase in overall sugar levels to corn kernels, but disadvantageously has only about one half of the total sugar content of “supersweet” corn varieties conferred by the Shrunken-2 (sh2) gene, which is significantly less desirable to corn consumers. Also disadvantageously, the conversion of sugar to starch in the corn kernels is comparatively rapid, generally resulting in a reduced or narrow harvest window before the sweetness (i.e., sugar levels) of the corn kernels deteriorates after the prime eating stage (at approximately 75% kernel moisture).
The sugary-1 (su1) gene, contrary to its name, therefore, does not generally result in exceptionally high levels of sugars. However, it does generally result in greatly increased levels of phytoglycogen or water soluble polysaccharides (WSP). (W. F. Tracey, In A. R. Hellauer (ED) Specialty Corns (CRC Press, Boca Raton, Fla.) 147-187 (1994).) Phytoglycogen and WSP tend to give the endosperm of the kernels of conventional su1 sweet corn varieties the smooth texture and creaminess characteristic of traditional sweet corn varieties. Mature endosperm of non-mutant corn generally contains approximately 2% WSP, whereas corn lines that are homozygous for the sugary-1 (su1) gene may contain up to approximately 35% WSP.
The mutant sugary-1 (su1) gene present in conventional varieties of sweet corn disadvantageously tends to result in a significantly reduced seed quality and use compared to field corn varietal counterparts.
Additional information about the recessive sugary-1 (su1) gene is present in M. G. James et al., “Characterization of the Maize Gene Sugary-1, a Determinant of Starch Composition in Kernels,” The Plant Cell, Vol. 7, 417-429 (1995); and D. Pan et al., “A Debranching Enzyme Deficiency in Endosperms of the Sugary-1 Mutants of Maize,” Plant Physiol. 74(2), 324-328 (1984).
Se1 (Sugary Enhancer-1) Mutant Gene
The sugary enhancer-1 (se1) mutant gene is a recessive modifier of the sugary-1 (su1) gene mutation. (J A Shultz et al., “Current Models for Starch Synthesis and the Sugary Enhancer 1 (se1) Mutation in Zea mays,” supra.). The effects of the sugary enhancer-1 (se1) mutant gene were originally observed in 111677a, a line derived from a three-way cross (Bolivia 1035X Illinois 44b X Illinois 442a). (J. W. Gonzales et al., “A New Inbred Line With High Sugar Content in Sweet Corn,” Hort Science 9, 79 (1974)). When homozygous, the sugary enhancer-1 (se1) allele increases total sugar in conventional sugary-1 (su1) sweet corn variety corn kernels to levels that are comparable to those in shrunken-2 (sh2) sweet corn variety corn kernels, and without a reduction in phytoglycogen content or an appreciable reduction in WSP levels. This allele also increases total sugar in conventional shrunken-2 (sh2) sweet corn variety corn kernels (also without a reduction in phytoglycogen content or an appreciable reduction in WSP levels).
The effects of the sugary enhancer-1 (se1) gene are corn kernel elevated total sugars, lighter color, and slow dry down, and were originally observed in an inbred corn line designated as IL1677a. It was only later that these effects were attributed to the sugary enhancer-1 (se1) gene. (Y. Tadmer et al., “RFLP Mapping of the Sugar Enhancer 1 Gene in Maize,” TAG Theoretical and Applied Genetics 91, 489-494 (1995); R A Brink, “Identity and Sources of a Sugary Enhancer Gene Located on the Long Arm of Chromosome 4 in Maize,” J. Heredity 82, 176 (1991).)
The sugary enhancer-1 (se1) gene confers a higher moisture content to sweet corn kernels during postharvest periods of time, and also maintains relatively increased levels of phytoglycogen during this time. Additional benefits of this gene are reduced kernel pericarp levels (i.e., thickness), rendering corn kernels with an improved tenderness, and elevated levels of the sugar maltose. (J E Ferguson et al., “Genetics of Sugary Enhancer (Se), an Independent Modifier of Sweet Corn (Su),” J. Heredity 69(6), 377-380 (1978).)
When both the sugary enhancer-1 (se1) gene and the sugary-1 (su1) gene are recessive, the sugary enhancer-1 (se1) gene very advantageously confers from about 1.5 to about 2 times more sucrose to corn kernels at their peak harvest maturity in comparison with sugary (su1) gene mutant corn kernels.
The sugary enhancer-1 (se1) gene locus is situated on the long arm of chromosome 2 in sweet corn. Identifiable variants of the sugary enhancer-1 (se1) gene are currently being evaluated and characterized.
Apparent difficulties in the genomic characterization of the sugary enhancer-1 (se1) gene have previously been encountered by scientists. Such difficulties are considered, in part, to be due to its rather difficult concomitant phenotypic measurement.
The enzymatic basis for the sugary enhancer-1 (se1) gene currently does not appear to be known, and the nucleotide sequence of the sugary enhancer-1 (se1) gene currently does not appear to be known, and is not present in the Maize Genetics and Genomics Database or GenBank database. However, the inheritance of the sugary enhancer-1 (se1) gene can be determined by those having ordinary skill in the art by following nearby molecular markers on chromosome 2, as is described herein. Such a determination may also be made for other mutant genes.
Additional information about the sugary enhancer-1 (se1) gene is present in D. R. La Bonte et al., “Sugary Enhancer (se) Gene Located on the Long Arm of Chromosome 4 in Maize (Zea mays L.), The Journal of Heredity 82, 176-178 (1991); and J. E. Ferguson et al., “The Genetics of Sugary Enhancer (se), an Independent Modifier of Sweet Corn,” The Journal of Heredity 69(6), 377-380 (1978).
Sh2 (Shrunken-2) Mutant Gene
In 1953, it was suggested that the mutant, recessive shrunken-2 (sh2) gene may have an application in the sweet corn industry. (J. R. Laughnan, “The Effects of sh2 Factor on Carbohydrate Reserves in the Mature Endosperm of Maize,” Genetics 38, 485-499 (1953).) Since then, a significant amount of research has been performed in connection with high sugar-content corn and the shrunken-2 (sh2) gene, which is located on the long arm of chromosome 3 in sweet corn, and encodes the large subunit of ADP-glucose pyrophosphorylase (AGP), a Class I enzyme. This enzyme is important in the conversion pathway of sucrose to starch.
The conventional shrunken-2 (sh2) class of sweet corns (designated as “supersweet”) comprises the vast majority of the U.S. commercial corn market. Mature dry shrunken-2 (sh2) variety corn kernels contain approximately twice the total sugar content, approximately ⅓ to ½ of the starch levels, and only trace levels of phytoglycogen, in comparison with conventional sugary-1 (su1) variety corn kernels. Shrunken-2 (sh2) type sweet corns generally result in dramatically reduced total carbohydrate levels at peak maturity, and express approximately two or more times the sucrose in comparison with conventional sugary-1 (su1) mutant sweet corn. Further, sugar retention at the post prime eating stage (i.e., during the period of time immediately following the prime eating stage) in these sweet corns is generally significantly extended relative to conventional sugary-1 (su1) and sugary enhancer-1 (se1) mutant sweet corns.
It has been determined that including the shrunken-2 (sh2) gene in the genetic makeup of corn advantageously has an effect of increasing the corn's total sugar content and, thus, sweetness. It has also been determined, however, that including this gene in the genetic makeup of corn very disadvantageously significantly lowers the corn's water soluble polysaccharide (WSP) content, reducing its endosperm content, and lowering its starch content (and its associated energy level). Disadvantageously, conventional shrunken-2 (sh2) varieties of corn generally lack the smooth and creamy texture of the conventional sugary-1 (su1) and sugary enhancer-1 (se1) mutant corn varieties as a result of such decreased levels of water soluble polysaccharides. (J A Shultz et al., “Current Models for Starch Synthesis and the Sugary Enhancer 1 (se1) Mutation in Zea mays,” supra.) Also very disadvantageously, as a result of a reduced starch content, and an associated reduced level of energy, conventional shrunken-2 (sh2) varieties of corn generally have a significantly reduced germination and seedling vigor, fitness and/or health during germination, seedling emergence from soil, and plant development and growth in comparison with the conventional sugary-1 (su1) and sugary enhancer-1 (se1) sweet corn varieties, resulting in light-weight, thin and spindly-looking, easily-damaged corn plants, which often readily die when confronted with environmental or other stresses, potentially causing corn growers to lose entire crops of corn, and the associated potential earnings from such crops. Shrunken-2 (sh2) varieties of sweet corn generally express a markedly collapsed kernel physical appearance at the dry seed stage, and this dry seed “shrunken” appearance, and corresponding reduced kernel starch reserves, tends to render a relatively diminished seedling emergence and vigor at planting time or during germination. In general, mechanical precision seeding and stand establishment are markedly more difficult due to the dry seed collapsed and “shrunken” physical appearance. The germination of such seeds can be problematic both in inbred production and in hybrid stands.
To improve vigor and germination, dent corn (a species of field corn) has been used as the genetic background for the shrunken-2 (sh2) gene. However, the dominant dent corn genes can very disadvantageously necessitate an isolation of the shrunken-2 hybrids from both field and sweet corn, and any foreign pollen can cause all of the corn kernels to be dent corn in character. In addition, shrunken-2 hybrids require spatial or temporal isolation from sugary-1 (su1) and sugary enhanced-1 (se1) sweet corn varieties due to the presence of dominant Su1Su1 alleles utilized in the shrunken-2 (sh2) “supersweet” sweet corn varieties.
Comparison of Sucrose Levels, Sugar Retention Abilities and Pericarp Levels
Comparisons of the sucrose levels, sugar retention (holding) abilities, pericarp levels and changes in pericarp levels of conventional sweet corn varieties containing the sugary-1 (su1) gene, the sugary enhancer-1 (se1) gene or the shrunken-2 (sh2) gene at the prime eating stage, or over a seven-day period, are shown in FIGS. 1-4, respectively. (Abbott and Cobb, Inc., Plant Protection No. 9600094 (1998).)
FIG. 1 provides a generalized comparison of representative endosperm sucrose levels for the conventional sugary-1 (su1), sugary enhancer-1 (se1) and shrunken-2 (sh2) genetic mutant lines at the prime eating stage (at a level of approximately 75% moisture). FIG. 1 shows that the sugary-1 (su1) line has the lowest level of sucrose (about 7.5%), followed by the sugary enhancer-1 (se1) line (about 12.5%), and then by the shrunken-2 (sh2) line, which has a much higher level of sucrose in comparison with the other two lines (about 27.5%).
FIG. 2 shows the relative endosperm sugar retention or “holding ability” at room temperature for the conventional sugary-1 (su1), sugary enhancer-1 (se1) and shrunken-2 (sh2) genetic mutant lines at the prime eating stage (at a level of approximately 75% moisture) over a seven day interval (Days 1-7). It shows representative changes in endosperm sucrose levels over time for the three different genetic types. FIG. 2 shows that the sugary-1 (su1) line has the lowest level of sucrose at all times during the 7-day period (ranging from about 4% on Day 1 to about 1% on Day 7), followed by the sugary enhancer-1 (se1) line (ranging from about 12% on Day 1 to about 2% on Day 7), and then by the shrunken-2 (sh2) line, which has a much higher level of sucrose over each of Days 1-7 in comparison with the other two lines (ranging from about 25% on Day 1 to about 13% on Day 7).
FIG. 3 provides a comparison of representative pericarp levels for the conventional sugary-1 (su1), sugary enhancer-1 (se1) and shrunken-2 (sh2) genetic mutant lines at the prime eating stage (at a level of approximately 75% moisture). FIG. 3 shows that the sugary enhancer-1 (se1) line has the lowest level of pericarp (about 0.75%), followed by the sugary-1 (su1) line (about 1.1%), and then by the shrunken-2 (sh2) line, which has a much higher level of pericarp in comparison with the other two lines (about 1.6%).
FIG. 4 shows the relative pericarp levels at room temperature for the conventional sugary-1 (su1), sugary enhancer-1 (se1) and shrunken-2 (sh2) genetic mutant lines at the prime eating stage (at a level of approximately 75% moisture) over a seven day interval (Days 1-7). It shows representative changes in pericarp levels over time for the three different genetic types.
FIG. 4 shows that the shrunken-2 (sh2) line generally has the lowest level of pericarp during the 7-day period (ranging from about 1.6% on Day 1 to about 2.1% on Day 7), followed by the sugary enhancer-1 (se1) line (ranging from about 1.1% on Day 1 to about 4.9% on Day 7) and the sugary-1 (su1) line (ranging from about 1.7% on Day 1 to about 4.9% on Day 7).
The major advantages of the higher sugar types of corn, i.e., sugary enhancer-1 (se1) and shrunken-2 (sh2) varieties, are their: (i) greater sweetness; (ii) longer harvest window of time; and (iii) longer shelf life. A higher initial sugar level, and a slower sugar loss at harvest, or during the period of time immediately following the prime eating stage, provide greater flexibility of harvest, and of handling conditions, and a longer shelf life for the corn.
Sh2-i (Shrunken-2i) Mutant Gene
U.S. Pat. No. 6,184,438 B1 describes an identification and characterization of a mutant form of the shrunken-2 (sh2) gene, designated as shrunken-2i (sh2-i). When present in maize plants, this mutant allele (and variants thereof) is stated to confer enhanced germination characteristics to these plants as compared to maize plants that express the sh2. This patent describes methods for transforming plants with this mutant allele (and variants), and plants that have this mutant allele incorporated into their genomes.
ADP-glucose pyrophosphorylase is a maize endosperm enzyme that is an important enzyme in the synthesis of starch, and catalyzes a conversion of ATP and α-glucose-1-phosphate to ADP-glucose and pyrophosphate. ADP-glucose arising from the action of this enzyme is the major donor of glucose for starch biosynthesis in plants. AGP enzymes have been isolated from plant photosynthetic and non-photosynthetic tissues, and is a heterotetramer that contains two different subunits.
Maize endosperm ADP-glucose pyrophosphorylase is composed of two dissimilar subunits that are encoded by two unlinked genes, shrunken-2 (sh2) and brittle-2 (bt2). (M. Bhave et al., “Identification and Molecular Characterization of Shrunken-2 cDNA Clones of Maize,” The Plant Cell 2:581-588 (1990); J. Bae et al., “Cloning and Characterization of the Brittle-2 Gene of Maize,” Maydica 35:317-322 (1990).) These genes encode the large subunit and the small subunit of this enzyme, respectively. The protein produced by the shrunken-2 gene has a predicted molecular weight of 57,179 Da. (J. Shaw et al., “Genomic Nucleotide Sequence of a Wild-Type Shrunken-2 Allele of Zea mays,” Plant Physiol. 98:1214-1216 (1992).)
Shrunken-2 (sh2) and brittle-2 (bt2) maize endosperm mutants generally have greatly reduced starch levels, which disadvantageously correspond with greatly reduced or deficient levels of AGP activity. Mutations of either gene have been shown to reduce AGP activity by about 95%. (C. Tsai et al., “Starch-Deficient Maize Mutant Lacking Adenosine Diphosphate Glucose Pyrophosphorylase Activity,” Science 151:341-343 (1966); D. Dickinson et al., “Presence of ADP-Glucose Pyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of Maize Endosperm,” Plant Physiol. 44:1058-1062 (1969).) It has also been observed that enzymatic activities increase with the dosage of functional wild type shrunken-2 (sh2) and brittle-2 (bt2) alleles, whereas mutant enzymes generally have altered kinetic properties. AGP appears to be the rate limiting step in starch biosynthesis in plants. (D. Stark et al., “Regulation of the Amount of Starch in Plant Tissues by ADP Glucose Pyrophosphorylase,” Science 258:287-292 (1992).)
The cloning and characterization of the genes encoding the AGP enzyme subunits have been reported for various plants, and include shrunken-2 (sh2) cDNA, shrunken-2 (sh2) genomic DNA, and brittle-2 (bt2) cDNA from maize, small subunit cDNA and genomic DNA from rice, small and large subunit cDNAs from spinach leaf, and potato tuber. In addition, cDNA clones have been isolated from wheat endosperm and leaf tissue and Arabidopsis thaliana leaf.
Gene splicing is essentially a two-step cleavage-ligation reaction that can produce molecular lesions in genes, such as the wildtype shrunken-2 (sh2) gene. The first step involves the cleavage at the 5′ splice site that leads to the formation of an intron lariat with the adenosine residue of the branch point sequence located upstream to the 3′ splice site. This is followed by the ligation of the exon and release of the intron lariat. (M. J. Moore et al., “Evidence of Two Active Sites in the Spliceosome Provided by Stereochemistry of Pre-mRNA Splicing,” Nature 365:364-368 (1993); M. J. Moore et al., “Splicing of Precursors to mRNAs by the Spliceosome” in The RNA World. 303-308 (R. Gesteland and J. Atkins, eds., Cold Spring Harbor Laboratory Press, 1993); P. A. Sharp, “Split Genes and RNA Splicing,” Cell 77:805-815 (1994); J. W. S. Brown, “Arabidopsis Intron Mutations and Pre-mRNA Splicing,” Plant J. 10(5):771-780 (1996); G. G. Simpson et al., “Mutation of Putative Branchpoint Consensus Sequences in Plant Introns Reduces Splicing Efficiency,” Plant J. 9(3):369-380 (1996); G. G. Simpson et al., “Splicing of Precursors to mRNA in Higher Plants: Mechanism, Regulation and Sub-nuclear Organization of the Spliceosomal Machinery,” Plant Mol. Biol. 32:1-41 (1996).) This set of events is carried out by pre-mRNA association with a conglomeration of small nuclear RNA (snRNAs) and nuclear proteins that forms a dynamic large ribonucleosome protein complex (a spliceosome). This fundamental process, common to all eukaryotic gene expression, can have a diverse impact on the regulation of gene expression. For example, imprecise or inaccurate pre-mRNA splicing often imparts a mutant phenotype, whereas alternative splicing is sometimes important in the regulation of gene expression. (C. F. Weil et al., “The Effects of Plant Transposable Element Insertion on Transcription Initiation and RNA Processing,” Annu. Rev. Plant Physiol. Plant Mol. Biol. 41:527-552 (1990); R. Nishihama et al., “Possible Involvement of Differential Splicing in Regulation of the Activity of Arabidopsis ANP1 that is Related to Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs),” Plant J. 12(1):39-48 (1997); M. Golovkin et al., “Structure and Expression of a Plant U1 snRNP 70K Gene: Alternative Splicing of U1 snRNP 70K Pre-mRNAs Produces Two Different Transcripts,” Plant Cell 8:1421-1435 (1996); J. Callis et al., “Introns Increase Gene Expression in Cultured Maize Cells,” Genes and Development 1: 1183-1200). There are structural/sequence differences that may differentiate plant introns from those of vertebrate and yeast introns. (G. J. Goodall et al., “The AU-Rich Sequences Present in the Introns of Plant Nuclear Pre-mRNAs Are Required for Splicing,” Cell 58:473-483 (1989); G. J. Goodall et al., “Different Effects of Intron Nucleotide Composition and Secondary Structure on Pre-mRNA Splicing in Monocot and Dicot Plants,” EMBO J. 10(9):2635-2644 (1991).) One feature that distinguishes plant introns from those of other organisms is their AU richness. This has been implicated to be essential for intron processing, and for a definition of the intron/exon junction. (H. Lou et al., “3′ Splice Site Selection in Dicot Plant Nuclei is Position Dependent,” Mol. Cell. Biol. 13(8):4485-4493 (1993); A. J. McCullough, “Factors Affecting Authentic 5′ Splice Site Selection in Plant Nuclei,” Mol. Cell. Biol. 13(3):1323-1331 (1993); J. C. Carle-Urioste et al., “In Vivo Analysis of Intron Processing using Splicing-Dependent Reporter Gene Assays,” Plant Mol. Biol. 26:1785-1795 (1994).) The requirement of an AU rich region appears to be more stringent in dicots in comparison with monocots, and some monocot introns are GC-rich.
The wildtype shrunken-2 (sh2) gene, the nucleotide sequence of which is shown in SEQ ID NO. 1, has 16 exons which, in term, are separated by 15 introns, as is shown in FIG. 5. This gene is estimated to be approximately 6000 base pairs long. One intron, in particular, is of significance for the sh2-i allele. This intron, designated “intron 2,” contains at least about 7,800 base pairs in the sh2-i gene.
In comparison with the wildtype shrunken-2 (sh2) gene, the mutant shrunken-2i (sh2-i) gene is characterized by a single base pair change at the end of intron 2, as is shown in FIGS. 5 and 9. The mutant polynucleotide comprises a substitution of the wild-type terminal base at the end of intron 2 from a G to another base, such as to A, C or T (and not the wild type G nucleotide), and preferably to A. For example, if the shrunken-2 gene of maize contains a G to A mutation of the terminal nucleotide of this intron, the result would be a change of the AG nucleotide sequence that is found at the terminus of this plant gene intron to an AA sequence at the 3′-terminus of this intron. In other words, the result is a molecular lesion of the sh2-i allele in which it has undergone a G to A mutation at the terminal base of intron 2 in the maize sh2 gene.
The mutant sh2-i allele (and variants thereof), when expressed in a plant, such as maize, provides the plant with enhanced growth characteristics, such as an enhanced germination, and an enhanced seedling, seed and/or plant vigor, in comparison with other sweet corn varieties, in combination with desirable consumer traits, such as sweetness.
Inbred sweet corn lines containing the mutant sh2-i allele (“sh2-i conversion inbreds”) generally demonstrate sweetness, and sugar levels, that are similar to conventional shrunken-2 (sh2) counterparts at the peak eating stage (at approximately 75% moisture). However, at a point in time just past the prime eating stage, mutant sh2-i inbred plant lines initiate a rapid acceleration of starch synthesis. This results in dry seed phenotypes that are physically significantly fuller and heavier than their shrunken-2 (sh2) counterparts and, to some degree, resemble a modified flint corn seed appearance. The net result is an overall enhancement of seed and seedling germination and vigor, and plant vigor, along with associated enhanced plant growth characteristics. This improved germination, and accelerated plant growth phenomenon, directly results in improved varietal crop yield potentials and consistencies.
Laboratory cold soil germination testing conducted by the present inventor comparing conventional shrunken-2 (sh2) hybrid maize varieties with maize varieties expressing the shrunken-2i (sh2-i) gene resulted in substantially stronger scores for the sh2-i hybrids. Table 2 below provides laboratory cold soil germination scores for corn hybrid near isogenic lines (NILs) designated as ACX 5137Y (not expressing the sh2-i allele) and ACX SS 7501Y (expressing the sh2-i allele). These are essentially comparisons of two maize hybrids differing only in the presence of the sh2-i allele. Scores represent means of three replicated tests of 100 kernels each.
TABLE 2Cold Soil Germination Scores for Isoline HybridsCold Soil GerminationIsoline Hybrid(% Germination)ACX 5137Y (not containing the sh2-i82allele in its genome)ACX SS 7501Y (containing the sh2-i97allele in its genome)
Similar laboratory results have been generated for numerous other hybrid comparisons between conventional shrunken-2 (sh2) hybrids containing, or not containing, the sh2-i allele. In addition, field germination and stand establishment data in Florida and California have substantiated laboratory data findings.
Organoleptic testing of numerous corn hybrid near isogenic lines (NILs) (conventional shrunken-2 (sh2) hybrid backcross conversions containing, and not containing, the sh2-i allele), however, disadvantageously yielded sweetness evaluation scores indicating a rapid starch buildup in the sh2-i hybrids, generally immediately following the prime eating stage. The sh2-i hybrids exhibited reduced sweetness within about 24 to about 48 hours immediately following the prime eating stage, with a corresponding significant sugar degradation and loss at about three days post prime eating stage.
FIG. 6 presents mean organoleptic averages for starch accumulation for conventional shrunken-2 (sh2) hybrid near isogenic lines (NILs) that do not contain the mutant shrunken-2i (sh2-i) allele in comparison with those for conventional shrunken-2 (sh2) hybrid corresponding near isogenic lines (NILs) that contain the mutant shrunken-2i (sh2-i) allele over time in Days 1-7 immediately following the prime eating stage (at a level of approximately 75% moisture). The organoleptic scores range from 1 (sweet with little or no starch taste) to 10 (very little sweetness with a considerable starch taste). FIG. 6 shows that the conventional shrunken-2 (sh2) hybrid near isogenic lines (NILs) that do not contain the mutant shrunken-2i (sh2-i) allele have significantly lower organoleptic scores (ranging from about 1 on Day 1 to about 4 on Day 7) in comparison with the conventional shrunken-2 (sh2) hybrid corresponding near isogenic lines (NILs) that contain the mutant shrunken-2i (sh2-i) allele (ranging from about 1.9 on Day 1 to about 7.9 on Day 7).
In essence, the incorporation of the shrunken-2i (sh2-i) allele into conventional sweet corn varieties is considered to be impractical due to a rapid accumulation of starch in the period of time immediately following the prime eating state, and the associated loss of holding ability and shelf life.